It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with messenger RNA (mRNA) or other intracellular RNA's that direct protein synthesis. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-stranded RNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding via Watson-Crick base pairs of the heterocyclic bases of oligonucleotides to RNA or DNA. Such base pairs are said to be complementary to one another.
Naturally-occurring events that provide for the disruption of the nucleic acid function, as discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989), are thought to be of two types. The first is hybridization arrest. This denotes the terminating even in which an oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller, P.S. and Ts'O, P.O.P. (1987) Anti-Cancer Drug Design, 2:117-128, and .alpha.-anomer oligonucleotides are the two most extensively studied antisense agents that are though to disrupt nucleic acid function by hybridization arrest.
In determining the extent of hybridization arrest of an oligonucleotide, the relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T.sub.m), a characteristic physical property of double helixes, denotes the temperature in degrees centigrade at which 50% helical versus coil (unhybridized) forms are present. T.sub.m is measured by using the UV spectrum to determine the formation and breakdown (melting) of hybridization. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently a reduction in UV absorption indicates a higher T.sub.m. The higher the T.sub.m, the greater the strength of the binding of the strands. Non-Watson-Crick base pairing has a strong destabilizing effect on the T.sub.m.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2'-deoxyribofuranosyl oligonucleotide hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides as antisense agents for diagnostics, research reagents and potential therapeutic purposes. This research has included the synthesis of oligonucleotides having various modification. Such modification have primarily been modifications of the phosphate links that connect the individual nucleosides of the oligonucleotide. Various phosphorothioates, phosphotriesters, phosphoramidates and alkyl phosphonates have been reported. Further research has been directed to replacement of the inter-nucleoside phosphates with other moieties such as carbamates, sulfonates, siloxanes and the formacetal group. Other modification have been effected wherein conjugate groups are attached to the nucleosides of the oligonucleotide via linking groups. Such conjugates include fluorescent dyes, intercalating agents, proteins, cross-linking agents, chain-cleaving agents and other groups including biotin and cholesterol. An extensive review discussing all of these modifications is that of Goodchild, J. (1990) Bioconjugate Chemistry, 1:165.
Since the heterocyclic bases of the nucieosides of an antisense oligonucleotide are necessary for the proper Watson/Crick binding of the antisense oligonucleotide to the target RNA or DNA, with the exception of cross-linking agents, little has been reported as to modification on the heterocyclic bases.
"Alpha" nucleosides have been used to form oligonucleotides having "alpha" sugars incorporated therein. In a like manner 2'-O-methylribonucleotides also have been used as precursor building blocks for oligonucleotides. U.S. Pat. No. 5,034,506 and PCT patent application PCT/US86/00544 suggest that the sugar portion of a nucleoside can be ring opened via oxidization and then ring closed by reactions with an amino or hydrazine group on an adjacent nucleoside. This links the nucleosides. Further, upon ring closure with the amino or hydrazine group, a new ring, a morpholine ring, is formed from the residue of the oxidized pentofuranose sugar ring of the nucleoside. PCT/US86/00544 also suggests that a linear amino acid based polymer might be used in place of a sugar-phosphate backbone to link heterocyclic bases together in an oligonucleotide-like linkage. Aside from these modifications, modification of the sugar moieties of the nucleosides of oligonucleotides is also little known.
In a further approach to modification of oligonucleotides both the sugar moieties and the phosphate linkers have been removed and replaced by a polymeric backbone. Utilizing this approach, heterocyclic bases have been tethered to various polymers including poly(N-vinyl), poly(methacryloxyethyl), poly(methacrylamide), poly(ethyleneimine) and poly(lysine). These types of compounds generally suffer from inappropriate spatial orientation of the heterocyclic bases for proper hybridization with a target RNA or DNA. A review of such polymeric compounds and the before noted "alpha" sugar containing oligonucleotides and 2'-O-methylribonucleotides is found in Uhlmann, E. and Peyman, A., (1990) Chemical Reviews, 90:543.
Recently oxetanocin and certain of its carbocyclic analogs have been studied as antiviral chemotherapeutic agents. These compounds incorporate an oxetane or a cyclobutane ring in place of the sugar moiety of a nucleoside. Cyclobut-A, i.e. (.+-.)-9-[(1.beta., 2.alpha., 3.beta.)-2,3-bis-(hydroxymethyl-1-cyclobutyl]adenine, and cyclobut-G, i.e. (.+-.)-9-[(1.beta., 3.alpha., 3.beta.)-2,3-bis(hydroxymethyl)-1-cyclobutyl]-guanine, were reported by Norbeck, D. W., Kern, E. Hayashi, S., Rosenbrook, W., Sham, H., Herrin, T., Plattner, J. J., Erickson, J., Clement, J., Swanson, R., Shipkowitz, N., Hardy, D., Marsh, K., Arnett, G., Shannon, W., Broder, S. and Mitsuya, H. (1990) J. Med. Chem., 33:1281. Further antiviral activity of these compounds was reported by Hayashi, S., Norbeck, D. W., Rosenbrook, W., Fine, R. L., Matsukura, M., Plattner, J. J., Broder, S. and Mitsuya, H. (1990) Antimicrobial Agents and Chemotherapy, 34:287. As reported in Hayashi et. al., both cyclobut-A and cyclobut-G exist as racemic mixtures of diastereomers. Also as reported by Hayashi et. al., the thymine, uracil and hypoxanthine analogs of cyclobut-A and cyclobut-G did not exhibit antiviral activity.
In an attempt to eliminate any effects that the racemic, diastereomeric 2,3- bis(hydroxymethyl)-1-cyclobutyl portion of cyclobut-A and cyclobut-G might have towards phosphorylation by kinases, non-diastereomeric 3,3-bis(hydroxymethyl)-1-cyclobutyl analogs of cyclobut-A and cyclobut-G were synthesized and reported by Boumchita, H., Legraverenc, M., Huel, C. and Bisagni, Eo (1990) J. Heterocyclic Chem. 27:1815.. However, contrary to the activity of cyclobut-A and cyclobut-G, the 3,3-bis(hydroxymethyl)-1-cyclobutyl analogs of adenine and guanine, i.e. 9-[3,3-bis(hydroxymethyl)cyclobut-1-yl]adenine and 9-[3,3-bis(hydroxymethyl)cyclobut-1-yl]guanine, respectively, were found to be devoid of antiviral activity.